GLUT4 vesicle dynamics in living 3T3 L1 adipocytes visualized with green-fluorescent protein

Abstract

Insulin stimulates glucose uptake into its target cells by a process which involves the translocation of the GLUT4 isoform of glucose transporter from an intracellular vesicular compartment(s) to the plasma membrane. The step(s) at which insulin acts in the vesicle trafficking pathway (e.g. vesicle movement or fusion with the plasma membrane) is not known. We expressed a green-fluorescent protein-GLUT4 (GFP-GLUT4) chimaera in 3T3 L1 adipocytes. The chimaera was expressed in vesicles located throughout the cytoplasm and also close to the plasma membrane. Insulin promoted a substantial translocation of GFP-GLUT4 to the plasma membrane. Time-lapse confocal microscopy demonstrated that the majority of GFP-GLUT4-containing vesicles in the basal state were relatively static, as if tethered (or attached) to an intracellular structure. A proportion (approx. 5%) of the vesicles spontaneously lost their tether, and were observed to move rapidly within the cell. Other vesicles appear to be tethered only on one edge and were observed in a rapid stretching motion. The data support a model in which GLUT4-containing vesicles are tightly tethered to an intracellular structure(s), and indicate that a primary site of insulin action must be to release these vesicles, allowing them to then translocate to and fuse with the plasma membrane.

Identification of P-Rex1 as a Novel Rac1-Guanine Nucleotide Exchange Factor (GEF) That Promotes Actin Remodeling and GLUT4 Protein Trafficking in Adipocytes

“…1B). The localization of GFP-GLUT4 is indistinguishable from endogenous GLUT4 in resting and insulin-treated adipocytes (48,49). No direct colocalization of P-Rex1 with GFP-GLUT4 was noted either in resting or insulin-stimulated adipocytes (Fig.…”

Rosiglitazone enhances glucose uptake in glomerular podocytes using the glucose transporter GLUT1

“…In addition, murine 3T3-L1 fibroblast clones were obtained from LGC Promochem (Teddington, UK). They were differentiated to adipocytes as previously described [23]. Finally, an immortalised human proximal tubular epithelial cell line (HK2) was used [24].…”

Role for the microtubule cytoskeleton in GLUT4 vesicle trafficking and in the regulation of insulin-stimulated glucose uptake

“…GLUT4 tagged at the N-terminus with green fluorescent protein (GFP) (GFP-GLUT4) resides in both the GSV compartment and in recycling endosomes, and translocates to the plasma membrane in response to insulin with characteristics expected of native GLUT4 [14,17]. We previously proposed that GLUT4 vesicles were apparently tethered to an intracellular anchor, and that a major role of the insulin-signalling pathway must be to release GLUT4 vesicles from this tether [17].…”